PROJECT SUMMARY Bacterial cells surround themselves with a peptidoglycan (PG) cell wall, an essential structure that resists changes in osmotic pressure and other environmental insults. To a certain degree, PG is also essential to humans as antibiotics target its destruction and fragments activate immune responses. The basic building blocks of PG have been known for over fifty years; however, the higher architectural features of this polymer and complete set of immunostimulating fragments remain unknown. We hypothesize that differences in overall PG structure and fragment generation are important for sensing pathogenic bacteria. The glycan of the PG is essential for immune recognition; study of this important structure has been hampered by a lack of tools to label and track the fate of PG carbohydrate precursors and the resultant polymer in living cells. Currently, researchers are limited to few carbohydrate probes and even fewer larger fragments. Chemical synthesis is laborious and challenging to even expert carbohydrate chemists. The goal of this U01 proposal is to develop a method to label the glycan of the PG in a range of microbes to facilitate identification, tracking, manipulation and analysis of the glycans derived from PG with their biological binding partners and determine their functions. We propose to utilize a metabolic labeling approach in which the necessary functionalized PG biosynthetic building blocks are synthesized, provided to the microbe and incorporated in the backbone of the polymer. This has not been done before as the synthesis of the UDP-sugar building blocks is challenging and the uptake and processing pathways for the free sugars are not widely distributed. To overcome this challenge we propose parallel approaches which utilize either chemoenzymatic synthesis or genetic engineering. The bacterial PG recycling enzymes, AmgK and MurU have relaxed substrate specificity for N-acetyl-muramic acid lactols, allowing the production of labeled UDP-PG precursors. In Aim One, a large-scale chemoenzymatic synthesis of a variety of UDP-PG derivatives will be optimized and these molecules will be provided to a variety of pathogenic and commensal microbes for subsequent PG incorporation. Kits will be developed to distribute these essential carbohydrates. For Aim Two tagged lactol substrates will be provided to cells whose genomes have been engineered to encode for AmgK and MurU. As Escherichia coli and Bacillus subtilis are amendable to this approach, this methodology will be extended to pathogens such as Helicobacter pylori (Hp) and Mycobacterium tuberculosis (Mtb) as well as commensal bacteria. Aim Three will showcase the utility of this method for immunologists and microbiologists: (1) glycan-containing immunostimulatory molecules from Mtb, and Hp will be tracked, sorted and identified; (2) Hp and Mtb's PG structural features related to pathogenesis and antibiotic susceptibility will be interrogated. This innovative carbohydrate metabolic labeling method for peptidoglycan will be an approachable yet powerful technique for biomedical researchers and a valuable addition to the Glycoscience Consortium.